Using Retron Recombineering for Efficient and Targeted Bacteriophage Genome Modification

Abstract

Bacteriophages have emerged as promising biological tools not only for combating infectious bacteria but also for facilitating the exploration of intra- and inter-protein interactions. However, conventional methods for editing phage genomes have been inefficient, involving tedious screening, rigorous counterselection, or in vitro construction processes. These limitations hinder our ability to modify phages effectively, thus restricting innovation in this field. In this study, we introduce a scalable approach for engineering phage genomes using recombitrons — modified bacterial retrons capable of generating recombineering donor ssDNA. Coupled with single-stranded binding and annealing proteins, these retrons facilitate the integration of donor DNA into target sequences within phage genomes or phagemids. Notably, this method enables efficient genome modifications across multiple phages and sites simultaneously without requiring counterselection. Furthermore, the process is continuous, allowing edits to accumulate in the phage genome over time, and multiplexable, enabling the introduction of distinct mutations from different editing hosts in a mixed culture. We demonstrate the utility of this approach by applying it to analyze intra-protein epistasis in the T7 phage tail fiber. Specifically, we generate host-specific/tolerant mutants in the gp17 tail fiber protein and employ a combinatorial variant library to infect E. coli mutants with truncated lipopolysaccharide coats — a condition often observed in multi-drug resistant bacteria. Our findings underscore the efficacy and scalability of retron recombineering for not only modifying endogenous phage proteins but also exogenous ones cloned into phagemids. This method significantly expands molecular biologists’ toolkit for genome editing and enhances our understanding of fundamental biological processes, including protein-protein interactions and epistasis.